Blood monocyte derived Neo-Hepatocytes as in vitro · 2008. 6. 16. · SULT1, Sulfotransferase 1A1...

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Blood monocyte derived Neo-Hepatocytes as in vitro

test system for drug-metabolism

S. Ehnert, A.K. Nussler, A. Lehmann and S. Dooley

Dept. of Medicine II, Faculty of Medicine at Mannheim, University of Heidelberg; Germany

[S.E. and S.D.]

Dept. of Traumatology, Technical University Munich, Germany [A.K.N. and current address

of S.E.]

Dept. General Surgery, University Medicine Berlin, Campus Virchow, Berlin, Germany

[A.L.]

DMD Fast Forward. Published on June 16, 2008 as doi:10.1124/dmd.108.020453

Copyright 2008 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on June 16, 2008 as DOI: 10.1124/dmd.108.020453

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Neo-Hepatocytes as alternative in vitro drug testing system

Corresponding author: Steven Dooley

Department of Medicine II, Gastroenterology and Hepatology,

University Hospital Mannheim, Germany

Theodor-Kutzer Ufer 1-3

68167 Mannheim

[email protected]

Phone: 0049-621-383-3768

Fax: 0049-621-383-1467

Number of text pages: 13 (total document 30 / 2 times page 2)

Number of tables: 5

Number of figures: 5

Number of references: 40

Abstract: 243 words

Introduction: 626 words including references

Discussion: 880 words including references

Abbreviations:

AHMC, 3-(2-(N,N-diethylamino)ethyl)-7-hydroxy-4-methylcoumarin

AMMC, 3-(2-N, N-diethyl-N-methylaminoethyl)-7-methoxy-4-methylcoumarin

BFC, 7-Benzyloxy-4-(trifluoromethyl)coumarin

CEC, 3-Cyano-7-ethoxycoumarin

CHC, 3-Cyano-7-hydroxycoumarin

CYP, Cytochrome P450

DBF, Dibenzylfluorescein

EFC, 7-Ethoxy-4-(trifluoromethyl)coumarin


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EPHX1, microsomal epoxide hydrolase 1

GST, glutathione-S-transferase

HC, 7-Hydroxycoumarin

HFC, 7-Hydroxy-4-(trifluoromethyl)coumarin

hrFGF4, human recombinant fibroblast growth factor 4

hrIL-3, human recombinant interleukin 3 (hrIL-3)

hrM-CSF, human recombinant macrophage colony-stimulating factor

KET, Ketoconazole

3-MC, 3-Methylcholanthrene

MCB, Monochlorobimane

MFC, 7-Methoxy-4-(trifluoromethyl)coumarin

n, number of replicates

N, number of individual experiments/donors

NAT1, N-acetyltransferase 1

NIF, Nifedipine

NMO, NAD(P)H menadione oxidoreductase 1

PBMC, peripheral blood monocytes

PCMO, programmable cell of monocytic origin

QUE, Quercetin dehydrate

RES, Resorufin

RIF, Rifampicin

SRB, Sulforhodamine B

SULT1, Sulfotransferase 1A1

UGT1A6, UDP-glucoronosyltransferase 1A6

VER, Verapamil


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Abstract

The gold standard for human drug metabolism studies is primary hepatocytes. However,

availability is limited by donor organ scarcity. Therefore, efforts have been made to provide

alternatives, e.g. the hepatocyte-like (NeoHep) cell type, which was generated from peripheral

blood monocytes (PBMCs). In this study, expression and activity of phase I and phase II

drug-metabolizing enzymes were investigated during trans-differentiation of NeoHep cells

and compared to primary human hepatocytes. Important drug metabolizing enzymes are

cytochrome P450 (CYP) iso-forms (1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2D6, 2E1, 3A4),

microsomal epoxide hydrolase 1 (EPHX1), glutathione-S-transferase (GST) A1 and M1, N-

acetyltransferase 1 (NAT1), NAD(P)H menadione oxidoreductase 1 (NMO1),

Sulfotransferase 1A1 (SULT1A1) and UDP-glucoronosyltransferase 1A6 (UGT1A6).

Monocytes and programmable cells of monocytic origin (PCMOs) expressed only a few of

the investigated enzymes. Throughout differentiation, NeoHep cells showed a continuously

increasing expression of all drug-metabolizing enzymes investigated, resulting in a stable

basal activity after approximately 15 days. Fluorescence based activity assays indicated that

NeoHep cells and primary hepatocytes have similar enzyme kinetics, although the basal

activities were significantly lower in NeoHep cells. Stimulation with 3-Methylcholanthrene

(3-MC) and Rifampicin (RIF) markedly increased CYP1A1/2 or CYP3A4 activities, which

could be selectively inhibited by Nifedipine (NIF), Verapamil (VER), Ketoconazole (KET)

and Quercetin (QUE). Our data reveal similarities in expression, activity, induction and

inhibition of drug-metabolizing enzymes between NeoHep cells and primary human

hepatocytes and hence suggest that NeoHep cells are useful as an alternative to human

hepatocytes for measuring bio-activation of substances.


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Introduction

Drug metabolism is a major determinant of drug clearance and most frequently responsible

for inter-individual differences in drug pharmacokinetics (Donato et al., 2004). Adverse

pharmacokinetics can result in altered or even inadequate responses to the drug, affecting its

use as therapeutic (Lin and Lu, 1997). In vitro screening became an invaluable tool to identify

the metabolic profile of drug candidates, potential drug interactions or the role of polymorphic

enzymes before starting clinical trials.

Drug metabolism in the liver can be divided into two phases (Williams, 1959). Phase I

metabolism adds a functional group (e.g. OH, SH or NH2) to the substrate by oxidative,

reductive and hydrolytic pathways. Phase II metabolic enzymes modify the newly introduced

functional group to O- and N-glucuronides, sulfate esters, various α-carboxy-amides, and S-

glutathionyl adducts in order to increase their polarity (Parkinson, 1996), making elimination

from the cells more rapid. Thus, hepatocytes mediate detoxification by activation of phase I

and II enzymatic pathways.

All members of the cytochrome P450 super-family, belonging to phase I drug-metabolizing

enzymes, can be identified by a highly conserved haem-thiolate functionality, responsible for

their catalytic mechanism (Nelson et al., 2004). Amino acid variations in their substrate

binding sites confer compound-, region- and stereo-selectivity of the enzymes (Guengerich

and MacDonald, 1990) representing the rate limiting step of drug biotransformation

processes. Experimental data suggest that most biotransformation of xenobiotics is done by

enzymes of the first three families (CYP1, 2 and 3) while other CYPs are involved in “house-

keeping” metabolism of endogenous molecules (Parkinson, 1996; Pelkonen et al., 1998).

To date, primary human hepatocyte cultures are the most powerful tool for in vitro studies

(Hewitt et al., 2007), although they have major limitations due to donor organ scarcity and

rapid cellular changes during culture (Guillouzo et al., 1993), resulting in a strong demand for


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alternative in vitro systems. The human hepatoma cell line HepG2, secreting low levels of

many plasma proteins characteristic for normal human liver cells (Knowles et al., 1980), is

best used to study induction of drug-metabolizing enzymes, as their basal expression is

significantly lower than in primary human hepatocytes (Rodriguez-Antona et al., 2002;

Wilkening et al., 2003) and strongly varies during culture time (Wilkening and Bader, 2003).

In recent years, numerous reports described the generation of hepatocytes or “hepatocyte-

like” cells from various types of extra-hepatic cells (Hengstler et al., 2005; Ruhnke et al.,

2005a; Ruhnke et al., 2005b; Nussler et al., 2006). In this study, we wanted to investigate the

usefulness of “hepatocyte-like” (NeoHep) cells, derived by trans-differentiation of peripheral

blood monocytes (PBMCs), as in vitro test system for drug screening purposes. Therefore, we

analyzed expression and activity of CYPs involved in xenobiotic metabolism with high

abundance in the liver, namely 1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2D6, 2E1 and 3A4 (Shimada

et al., 1994; Rendic and Di Carlo, 1997; Hewitt et al., 2007). There exist various modes of

regulation, defining the relative abundance and activity of each CYP iso-form, that include

induction (CYP1A1, 1A2, 2A6, 2E1, 2C and 3A4), inhibition (all CYPs) and genetic

polymorphism (CYP2A6, 2C9, 2C19 and 2D6) (Rendic and Di Carlo, 1997), which are

important to ascertain to minimize potential drug-drug interactions when developing drug

candidates (Lin and Lu, 1998; Hewitt et al., 2007). Therefore, we investigated the effect of

model inducers (3-MC and RIF) and inhibitors (NIF, VER, KET and QUE) on CYP

expression and activity.

Most reports only describe the expression of CYPs and neglect the expression of phase II

enzymes, which are also important for activation and detoxification of many xenobiotics

(Cantelli-Forti et al., 1998). To obtain a more complete view about the drug-metabolizing

potential of NeoHep cells, expression and activity of phase II enzymes, EPHX1, GST A1 and

M1, NAT1, NMO1, SULT1A1 and UGT1A6 were determined in the present work.


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Methods

Chemicals

The following chemicals were used: hrFGF-4, hrIL-3 and hrM-CSF (R&D Systems,

Minneapolis, USA); AHMC, AMMC, anti-β-actin antibody, BFC, CEC, CHC, Coumarin,

DBF, EFC, HC, HFC, Histopaque-1077, KET, MCB, 2-Mercaptoethanol, 3-MC, MFC, NIF,

QUE, RES, RIF, SRB, Trizol, VER and William’s Medium E (Sigma, München, Germany);

FBS (EC approved – S. America), L-Glutamine, human serum type AB, Penicillin/

Streptomycin and RPMI 1640 Medium (Cambrex, Taufkirchen, Germany). β-

Glucuronidase/Arylsulfatase mix (Roche, Mannheim, Germany); anti-CYP2C8/9/19, -

CYP2E1, -CYP3A4 antibodies (CHEMICON, Chandlers Ford, UK); anti-CYP1A1/2, -

CYP2A/B6 and -CYP2D6 antibodies (Santa Cruz Biotechnology, Santa Cruz, USA);

horseradish peroxidase-conjugated secondary antibodies (Cell Signaling, Danvers, USA);

NuPage Bis-Tris gels (Invitrogen; Karlsruhe, Germany).

Generation of programmable cells of monocytic origin (PCMOs)

PCMOs were generated from peripheral blood of healthy volunteers as described (Ruhnke et

al., 2005a; Ruhnke et al., 2005b). Withdrawal of blood was approved by the local ethics

committee of the Medical Faculty at Mannheim, University of Heidelberg (Proposal No

164/05). Briefly, peripheral blood monocytes (PBMCs) were isolated by density gradient

centrifugation (Histopaque-1077). The resulting mononuclear cell fraction was allowed to

adhere to tissue culture plastic (1.0 * 106 cells/cm2) for 2 h in RPMI 1640 medium (10 %

human AB serum, 2 mM L-Glutamine, 100 U/ml Penicillin and 100 µg/ml Streptomycin).

Non-adherent cells were removed by aspiration and remaining cells were cultured for 6 days

in RPMI 1640 based medium (see above) supplemented with 5 ng/ml hrM-CSF, 0.4 ng/ml

hrIL-3 and 0.1 mM 2-mercaptoethanol.


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Generation and culture of NeoHep cells

For differentiation into NeoHep cells, PCMOs (day 6) were cultured in hepatocyte

conditioning medium (RPMI 1640, 10 % FBS, 2 mM L-Glutamine, 100 U/ml Penicillin, 100

µg/ml Streptomycin, 3 ng/ml hrFGF-4) as reported (Ruhnke et al., 2005a; Ruhnke et al.,

2005b). Culture medium was replaced every third day.

Isolation of primary human hepatocytes

Human liver tissue was obtained according to the institutional guidelines from liver resections

of tumor patients with primary or secondary liver tumors. Hepatocytes were isolated by a two-

step collagenase perfusion technique followed by a Percoll gradient centrifuge for

purification, as described (Dorko et al., 1994). Hepatocyte viability was consistently above 90

%, as assessed by the trypan blue exclusion test. Freshly harvested hepatocytes were cultured

on rat-tail collagen-coated culture plates in Williams’ E medium (10 % FCS, 1 M Insulin, 15

mM HEPES, 1.4 M Hydrocortisone, 100 U/ml Penicillin and 100 mg/ml Streptomycin).

Treatment of cells

Prior to all experiments, the cells were serum starved overnight in William’s Medium E (2

mM L-Glutamine). Treatment with the different substances was performed in serum free

medium for indicated times and concentrations. Control conditions included cells maintained

for the same period in serum-free medium supplemented with the solvent chemical.

Conventional RT-PCR

Total cellular RNA was isolated using Trizol reagent according to the manufacturers’

protocol. First-strand cDNA was synthesized from 1 µg total RNA using the QuantiTect

reverse transcription kit (Qiagen, Hilden, Germany). Primer sequences and the corresponding

annealing temperatures are summarized in Table 1. Appropriate cDNA dilutions and PCR


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cycling numbers were determined for each gene to ensure that the PCR did not reach

saturation. PCR products resolved by gel electrophoresis in a 1.5 % (w/v) agarose gel (in

TBE) were visualized by ethidiumbromide.

Western blot analysis

Cells were lysed in ice cold RIPA buffer (50 mM TRIS; 250 mM NaCl; 2 % Nonidet P40; 2.5

mM EDTA; 0.1 % SDS; 0.5 % DOC; protease inhibitor and 1.0 % phosphatase inhibitor, pH

7.2) as described (Wiercinska et al., 2006). Protein concentration was measured with the DC

Protein Assay Kit (BioRad, Munich, Germany). Total protein lysates were separated by SDS

PAGE using NuPAGE Bis-Tris gels and transferred to nitrocellulose membranes (VWR,

Darmstadt, Germany). Immunoblotting proceeded as described (Weng et al., 2007).

Cytochrome P450 activity assays

Fluorescence-based P450 assays, were performed by incubation of intact cells (in 96-well-

plates) with selected substrates as reported (Donato et al., 2004). Briefly, 100 µl reaction

buffer (1 mM Na2HPO4; 137 mM NaCl; 5 mM KCl; 0.5 mM MgCl2; 2 mM CaCl2; 10 mM O-

(+)-Glucose; 10 mM HEPES; pH 7.4) containing the appropriate amount of fluorogenic

substrate were added to each well. After incubation at 37 °C, supernatants were transferred to

white/black 96-well-plates and cells were fixed for protein quantification by SRB staining.

Potential metabolite conjugates formed were hydrolyzed by incubation of supernatants with

β-Glucuronidase/Arylsulfatase (150 Fishman units/ml; 1200 Roy units/ml) for 2 h at 37.0 °C.

Samples were diluted (1:4) with the appropriate quenching solution. Formation of fluorescent

metabolite was quantified by means of a Fluoroskan Ascent fluorescence microplate reader

(ThermoLabsystems, Egelsbach, Germany). Results are given as picomoles of metabolite

formed per minute normalized to total protein content. Experimental conditions are

summarized in Table 2. Methanol fixed cells were used for background subtraction.


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Phase II enzyme activity assays

Cells, cultured in 96-well.plates, were incubated at 37 °C with 100 µl reaction buffer (1 mM

Na2HPO4; 137 mM NaCl; 5 mM KCl; 0.5 mM MgCl2; 2 mM CaCl2; 10 mM O-(+)-Glucose;

10 mM HEPES; pH 7.4), containing the appropriate amount of fluorescent substrate (products

from CYP assays). After 15, 30, 45, 60, 90 and 120 min the remaining fluorescent signal in

the supernatant (Fluoroskan Ascent fluorescence microplate reader ) was determined and cells

were fixed for protein quantification by SRB staining. Results are given as nanomoles of

fluorescent substrate reduced per minute normalized to total protein content. Methanol fixed

cells were used as negative control (background subtraction).

SRB staining

SRB staining was performed as reported (Skehan et al., 1990). Briefly, cells were covered

with ice cold fixation buffer (95 % Ethanol, 5 % acetic acid) and kept at -20 °C for 1 h. Fixed

cells were stained with 0.4 % SRB (w/v) in 1 % acetic acid for 30 min. Unbound dye was

removed by washing with 1 % acetic acid. Bound SRB was resolved in 10 mM un-buffered

TRIS solution and ODs at 565 nm (SRB) and 690 nm (back-ground) were determined. From

the ODs, we calculated the total protein content with the standard curve (y = 1.0305x –

1.6519; R2 = 0.9868), obtained by plotting the OD from SRB staining vs. total protein

contents measured with the DC Protein Assay Kit.

Statistics

Results are expressed as mean ± standard error of the mean (s.e.m.). Curve fitting was

performed using the GraphPad Prism software (El Camino Real, USA), which allowed

determination of R2, kinetics parameters and EC50 values. Results were analyzed by analysis

of variance (ANOVA) followed by paired comparison (Bonferroni), as appropriate. P < 0.05

was taken as the minimum level of significance.


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Results

Expression of phase I and II drug metabolizing enzymes during trans-differentiation of

NeoHep cells

Conventional RT-PCR was performed in order to assess mRNA levels of human phase I drug

metabolizing enzymes, CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9,

CYP2D6, CYP2E1 and CYP3A4 (Figure 1a) and human phase II drug metabolizing enzymes,

EPHX1, GST A1 and M1, NAT1, NMO1, SULT1 and UGT 1A6 (Figure 1c) during trans-

differentiation of NeoHep cells.

NeoHep cells as well as primary hepatocytes displayed variations in mRNA levels between

different donors. To prevent such donor variations, cDNAs from 5 individual experiments

were pooled. Overall, expression of drug metabolizing enzymes was present, but much lower

in NeoHep cells compared to human hepatocytes. In monocytes and PCMOs, except CYP1A1

and CYP2E1, no other CYP iso-forms were expressed, whereas NeoHep cells showed mRNA

and protein of all CYP iso-forms investigated. Compared to human hepatocytes (day 4 of

culture) CYP mRNA levels in NeoHep cells were low (1A1 ~26 %, 1A2 ~38 %, 2A6 ~20 %,

2B6 ~32 %, 2C8 ~19 %, 2C9 ~27 %, 2D6 ~41 %, 2E1 ~25 % and 3A4 ~17 % of human

hepatocytes). CYP2A6 and CYP3A4 could not be detected in every sample investigated.

Noteworthy, PCR for CYP1A2 gave, in addition to the product with the expected size, present

in NeoHep cells and human hepatocytes, a smaller not yet identified product (*) for

monocytes, PCMOs and NeoHep cells, which was lacking in hepatocytes. Protein expression

analysis by Western blot, using pooled samples (N=5) of monocytes, PCMOs, NeoHep cells

and human hepatocytes, confirmed the PCR results (Figure 1b).

All phase II drug metabolizing enzymes investigated were expressed (mRNA) in NeoHep

cells (Figure 1c). Similar to CYP iso-forms, phase II enzyme expression was generally lower

in NeoHep cells (EPHX1 ~68 %, GST A1 ~87 %, GST M1 ~72 %, NAT1 ~66 %, NMO1 ~66


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%, SULT1 ~73 %, UGT1A6 ~29 %) compared to human hepatocytes. Most phase II enzymes

except GST A1 and UGT 1A6, which were exclusively expressed in NeoHep cells and

primary hepatocytes, were also expressed in monocytes and PCMOs.

Incubation with CYP isozyme selective substrates leads to comparable metabolite

release over time in NeoHep cells and human hepatocytes

Enzyme kinetic experiments were performed with substrates that are iso-zyme selective for

CYP1A1/2, CYP2A6, CYP2B6, CYP2C8/9, CYP2D6, CYP2E1 and CYP3A4 in both

NeoHep cells (N=4; n=4) and human hepatocytes (N=4; n=4) (Figure 2a-g). Cells were

incubated for 0-120 minutes with the appropriate substrates. Metabolite conjugates formed

were hydrolyzed for 2 h at 37.0 ° C with β-Glucuronidase/ Arylsulfatase. The amount of

metabolite formed was normalized to total protein content and plotted vs. the incubation time.

The correlation coefficients (R2) from curve fitting are summarized in Table 3. RT-PCR and

Western blot indicated lower expression of CYP iso-forms in NeoHep cells compared to

human hepatocytes (Figure 1a, b), thus it was not surprising that the plateau values (Table 3)

were lower in NeoHep cells. However, the time-point when the plateau is half reached

(t1/2Plateau; Table 3) are comparable between both cell types. Based on these results, substrate

incubation times for all further experiments were determined to be between t1/2Plateau and tPlateau

(Table 2) not to measure product release during steady state (plateau) conditions.

NeoHep cells and human hepatocytes display similar phase II drug metabolism

Since CYP activities were measured in intact cells, metabolites formed by CYP iso-forms are

continuously processed by various phase II drug metabolizing enzymes. Phase II activities in

human hepatocytes (N=4; n=4) and NeoHep cells (N=4; n=4) are presented as reduction

(conjugation) of metabolites AHMC, CHC, HC, HFC, Fluoresceine and Resorufin (Figure 3a-

f). Furthermore, GST activity was measured by conjugation of MCB (Figure 3g).


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The amount of metabolite degraded was normalized to total protein content and plotted vs. the

corresponding incubation times. Correlation coefficients (R2) and half life times (t1/2) obtained

from curve fitting were comparable between NeoHep cells and human hepatocytes (Table 4).

RT-PCR results indicated that expression of phase II drug metabolizing enzymes is lower in

NeoHep cells when compared to freshly isolated human hepatocytes (Figure 1c). This is

supported by enhanced degradation of metabolites in human hepatocytes given by lower

plateau phase values for CHC, HC and HFC, whereas those for Fluoresceine degradation were

comparable between both cell types.

Possible metabolite conjugates formed were hydrolyzed by incubating supernatants with β-

Glucuronidase/Arylsulfatase (150 Fishman units/ml and 1200 Roy units/ml) as described

(Donato et al., 2004). Kinetics for CHC, HC, HFC and Fluoresceine “back-reaction” were

measured and an incubation time of 2 h at 37 °C was sufficient to hydrolyze all formed

metabolite conjugates (data not shown).

Basal CYP activities increase during differentiation of NeoHep cells

After having defined reaction conditions for CYP1A1/2, CYP2A6, CYP2B6, CYP2C8/9,

CYP2D6, CYP2E1 and CYP3A4, basal CYP activities were measured during trans-

differentiation of monocytes to NeoHep cells and compared to freshly isolated human

hepatocytes (Figure 4a-g). Monocytes (N=11; n=4) and PCMOs (N=11; n=4) showed no

significant activities for the investigated CYP iso-forms. CYP activities of NeoHep cells

(N=12; n=4; measured every 5th day in culture) increased during the first 15 days, then

reaching a stable level. Compared to human hepatocytes (N=10; n=4), maximal CYP

activities of NeoHep cells were significantly lower, which is in line with results from RT-PCR

and Western blot (Figure 1a, b).


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Model inducers increase CYP1A1/2 and CYP3A4 expression and activity in NeoHep

cells and human hepatocytes

Of the six different CYP iso-zymes studied, CYP1A1/2 and CYP3A4 are known to be highly

inducible by xenobiotics. 3-MC and RIF were selected as specific inducers for CYP1A1/2 and

CYP3A4, respectively. NeoHep cells (N=12; n=3) and human hepatocytes (N=4; n=4) were

incubated with 25 µm 3-MC or RIF. DMSO was used as solvent control. After 72 h the

residual stimulation medium was washed off the cells and CYP1A1/2 and CYP3A4 activities

were determined. Although basal CYP1A1/2 and CYP3A4 activities were reduced during the

3 days incubation time in primary hepatocytes, values were still slightly, but consistently,

higher than in NeoHep cells, which kept the basal CYP1A1/2 and CYP3A4 activity levels.

Therefore, induction is given as fold of control. In both cell types CYP1A1/2 activity was

significantly increased with 3-MC (Hep 3.78 ± 1.38; NeoHep 2.47 ± 0.89), but not with RIF

(Figure 5a), whereas CYP3A4 activity was increased with RIF (Hep 3.32 ± 0.97; NeoHep

2.89 ± 0.78), but not with 3-MC (Figure 5b). From the same experiment protein lysates were

assessed by Western blot, indicating that expression levels of CYP1A1/2 and CYP3A4 were

selectively increased by 3-MC and RIF, respectively (Figure 5c), which confirms results from

activity measurements.

Effect of model inhibitors on CYP1A1/2 and CYP3A4 activity is comparable in NeoHep

cells and human hepatocytes

NIF, VER, KET and QUE are model inhibitors for various CYP iso-forms. Since basal levels

of all CYP iso-forms were significantly lower in NeoHep cells compared to freshly isolated

human hepatocytes, effects (EC50) of these CYP inhibitors on induced CYP1A1/2 and

CYP3A4 activities were investigated in both cell types. Human hepatocytes (N=2; n=2) and

NeoHep cells (N=4; n=2) were treated with 25 µM 3-MC or RIF. DMSO was used as solvent

control. After 72 h the stimulation medium was removed and the cells were pre-incubated for


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15 min with different concentrations of Nifedipine, Verapamil, Ketoconazol or Quercetin,

before measuring CYP1A1/2 and CYP3A4 activities in the presence of the inhibitors. The

corresponding correlation coefficients (R2) and EC50 values are summarized in Table 5.

Discussion

In the present study NeoHep cells were generated from PBMCs donated by healthy volunteers

in order to evaluate their potential as an alternative in vitro test system for drug screening.

Secretion of urea, glucose and production of alanine-amino-transferase (ALT) as well as

aspartate-amino-transferase (AST) confirmed earlier observations (Ruhnke et al., 2005a),

ensuring the quality of our NeoHep cells. We compared expression and activity of phase I

drug-metabolizing enzymes of NeoHep cells and primary human hepatocytes, which comprise

over 90 % of drug oxidation in humans, namely CYP1A2 (4-6 %), 2A6 (2 %), 2B6 (25 %),

2C9 (10-11 %), 2D6 (25 %), 2E1 (2-5 %) and 3A4 (52 %) (Shimada et al., 1994). In contrast

to PCMOs, which only express CYP2E1, expression of all CYP iso-forms were increased

during differentiation of NeoHep cells, however, remained much lower compared to isolated

human hepatocytes. Several reports show a gradual decrease in CYP expression over time in

primary human hepatocytes (LeCluyse, 2001; Rodriguez-Antona et al., 2002; Gomez-Lechon

et al., 2004), limiting their use for long term studies. To overcome this problem hepatocytes

have been cultured between two layers of collagen (Kern et al., 1997; Tuschl and Mueller,

2006) or in the presence of appropriate cocktails of inducers (Pichard-Garcia et al., 2002).

Therefore, for in vitro screening of new substances or to predict their possible toxicology,

there are many hepatocyte-like cell lines in use. However, most of these cell lines have major

limitations, e.g. reduced expression of xenosensors, leading to differences in drug metabolism

(Hewitt et al., 2007), as well as incomplete and very low expression levels of drug

metabolizing enzymes (Wilkening et al., 2003, Donato et al., 2008). HepG2 and Hep1c1c7


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cells, for example, display strongly down-regulated expression of the pregnane X receptor

(PXR) and the constitutive androstane receptor (CAR) (Pascussi et al., 2001) strongly

involved in the regulation of CYP3A4. Increased CYP3A4 expression in NeoHep cells by

RIF indicates a sufficient expression level of both receptors in this cell type (Gerbal-Chaloin

et al., 2006). Similar to HepG2 and Hepa1c1c7 cells, the arylhydrocarbon receptor (Ahr),

strongly involved in the regulation of CYP1A iso-enzymes, is expected to be expressed in

NeoHep cells, since CYP1A1 and CYP1A2 were inducible with 3-MC (Pascussi et al., 2001).

A detailed analysis, indicated that CYP expression levels in HepG2 cells varied heavily

during culture, leading to strongly variable passage dependent results (Wilkening and Bader,

2003). In contrast, CYP activity levels of NeoHep cells remained constant for several days.

Similar to hepatoma cell lines, the basal CYP activities in NeoHep cells were also very low,

possibly limiting their use for inhibition experiments. Nevertheless, we were able to show

(competitive) inhibition of CYP1A1/2 and CYP3A4 activity induced by 3-MC and RIF, using

NIF, VER, KET and QUE. The resulting EC50 values in NeoHep cells were lower than in

human hepatocytes. This might be explained by relatively low CYP baseline levels in

NeoHep cells or nonspecific binding of substrate to cell membranes and albumin.

Phase II drug metabolizing enzymes were only slightly reduced in NeoHep cells compared to

primary human hepatocytes (Ruhnke et al., 2005a). While CYP iso-form activities are

reduced in the presence of HGF, phase II enzymes UGT and GST are not regulated by this

growth factor (Donato et al., 1998). In contrast to CYP iso-forms, almost all phase II drug-

metabolizing enzymes, except GST A1 and UGT 1A6, were already expressed in monocytes

and PCMOs and their expression level did not increase significantly during differentiation to

NeoHep cells. Thus, it was not surprising that degradation kinetics of AHMC, CHC, HC,

HFC, Fluoresceine and Resorufin were comparable between NeoHep cells and primary

hepatocytes, with higher plateau values for NeoHep cells. Similar results were observed for

GST activity, represented by MCB conjugation. These results are supported by an earlier


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observation (Ruhnke et al., 2005b) showing that UGT activity, measured by 4-

methylumbelliferone conjugation, is comparable between both NeoHeps cells and primary

human hepatocytes.

Phenotypic as well as genotypic differences in the expression of drug-metabolising enzymes

are the main causes for high inter-individual metabolic variations (Hewitt et al., 2007), not

taking into consideration non-genetic factors like smoking, age, diet, hormonal status,

environmental chemicals and disease state (Shah, 2005; Singh, 2006). There are many

polymorphisms reported, which may lead to complete inactivation, decreased activity or

altered substrate specificity of CYPs (Ingelman-Sundberg, 2005). In line with this, expression

and activity of drug-metabolizing enzymes varied strongly among hepatocytes from different

donors (Rodriguez-Antona et al., 2001). Similar donor-dependent variations were observed

for NeoHep cells. Donor variations in the response to different inducers, further complicates

predictions of therapeutic doses, which can lead to severe side-effects. In case of the anti-

coagulant Warfarin, certain variants of CYP2C9 and VKORC1 (vitamin K epoxide reductase

complex, subunit 1) require lower doses of warfarin than average to obtain the same

therapeutic effect, and are more likely to suffer from bleeding complications at standard doses

(Jones, 2007). This underlines the need for a “personalized medicine”, that comprises

screening of different therapeutic approaches in vitro before applying it to the patient (Singh,

2006). Though direct comparison is still missing, NeoHep cells display a similar individual

variability in CYP expression and activity as seen in human hepatocytes. Since NeoHep cells

can be generated from blood samples, they represent a promising tool for personalized

therapeutic screenings in the future.

Acknowledgements

We appreciate excellent technical assistance from A. Müller.


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Footnotes

a) The present work was financially supported by the Dietmar Hopp Foundation, the

Federal Ministry of Research (BMBF 0313081B / HepatoSys) and Fresenius Biotech GmbH.

b) Steven Dooley

Department of Medicine II, Gastroenterology and Hepatology, University Hospital

Mannheim

Theodor-Kutzer Ufer 1-3

68167 Mannheim, Germany

Email: [email protected]

Phone: 0049-621-383-4983


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Legends for figures

Figure 1: NeoHep cells express phase I and II drug metabolizing enzymes. RT-PCR and

Western blot were performed with pooled samples from 5 individual preparations of

monocytes (mono), programmable-cells of monocytic origin (PCMO), NeoHep cells from

healthy controls and human hepatocytes (Heps). cDNA template and total protein contents

loaded were adjusted not to reach saturation of signal (a) RT-PCR for basal mRNA levels of

human CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2D6, CYP2E1 and

CYP3A4. * Unexpected PCR product, which is not present in Heps. (b) Western blot showing

basal protein levels of CYP1A1/2, CYP2A/B6, CYP2C9, CYP2D6, CYP2E1 and CYP3A4.

(c) RT-PCR showing basal mRNA levels of the human phase II enzymes, EPHX1, GST A1,

GST M1, NAT1, NMO1, SULT1A1 and UGT1A6 (a-c) β-actin was used as loading control.

Figure 2: NeoHep cells display similar Cytochrome P450 metabolite release over time as

human hepatocytes. NeoHep cells (N=4; n=4; straight line) and human hepatocytes (N=4;

n=4; dotted line) were incubated for 0-120 min with the individual substrates. After

hydrolysis of metabolite conjugates with β-Glucuronidase/Arylsulfatase, the amount of

metabolite formed was normalized to total protein content and plotted vs the corresponding

incubation times. Curve fitting for kinetics of (a) CYP1A1/2, (b) CYP2A6, (c) CYP2B6, (d)

CYP2C8/9, (e) CYP2D6, (f) CYP2E1 and (g) CYP3A4 was done using the GraphPad Prism

software. Background signal was determined by incubation of methanol fixed cells and

subtracted from each measurement value.

Figure 3: Reduction (conjugation) of metabolites as measure of active phase II drug

metabolism. NeoHep cells (N=4; n=4; straight line) and human hepatocytes (N=4; n=4;

dotted line) were incubated for 0-120 min with a defined concentration of the metabolites

formed during CYP enzyme assays. Reduction (conjugation) of (a) AHMC (ex/em = 390/460

nm), (b) CHC (ex/em = 390/460 nm), (c) HC (ex/em = 355/460 nm), (d) HFC (ex/em =


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390/520 nm) (e) Fluoresceine (ex/em = 485/538 nm) and (f) Resorufin (ex/em = 530/590 nm)

was determined by the decrease in fluorescent signal. The amount of substrate degraded was

normalized to total protein content and plotted vs the corresponding incubation times.

Background signal was determined by incubation of methanol fixed cells and subtracted from

each measurement value. Curve fitting was done using the GraphPad Prism software.

Figure 4: Basal Cytochrome P450 activities during trans-differentiation of NeoHep cells.

Basal CYP enzyme activities in monocytes (mono; N=11; n=4), PCMOs (N=11; n=4),

NeoHep cells (N=12; n=4) at days 5, 10, 15 and 20 of differentiation and human hepatocytes

(Hep; N=10; n=4). Cytochrome P450 iso-forms measured were (a) CYP1A1/2, (b) CYP2A6,

(c) CYP2B6, (d) CYP2C8/9, (e) CYP2D6, (f) CYP2E1 and (g) CYP3A4. Activity was given

as pmol of metabolite/min/mg protein. * p < 0.05, ** p < 0.01; *** p < 0.001 of values being

significantly greater than 0.

Figure 5: Induction of CYP1A1/2 and CYP3A4 in NeoHep cells and human hepatocytes

with rifampicin and 3-methylcholanthrene NeoHep cells (N=12; n=3; filled bars) and

human hepatocytes (N=4; n=4; empty bars) were incubated for 72 h with 25 µM 3-MC or 25

µM rifampicin (RIF). DMSO (0.1 %) was used as solvent control. (a) CYP1A1/2 activity and

(b) CYP3A4 activity was determined and fold induction by 3-MC and RIF was calculated. (c)

Protein lysates were taken to confirm increased expression of CYP1A1/2 and CYP3A4 by

Western blot in both cell types. 30 µg total protein lysate was loaded per lane. β-actin was

used as loading control. ** p < 0.01; *** p < 0.001.


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Tables

Table 1: PCR conditions

Accession Sequence 5' to 3' Amplicon Tm

[NM_] Forward Primer Reverse Primer bp [°C]

CYP1A1 000499 TTC GTC CCC TTC ACC ATC CTG AAT TCC ACC CGT TGC 302 55

CYP1A2 000761 TCG TAA ACC AGT GGC AGG T GGT CAG GTC GAC TTT CAC G 254 60

CYP2A6 000762 CAA CCA GCG CAC GCT GGA TC CCA GCA TAG GGT ACA CTT CG 423 60

CYP2B6 000767 ATG GGG CAC TGA AAA AGA CTG A AGA GGC GGG GAC ACT GAA TGA C 283 62

CYP2C8 000770 CAT TAC TGA CTT CCG TGC TAC AT CTC CTG CAC AAA TTC GTT TTC C 147 60

CYP2C9 000771 CTG GAT GAA GGT GGC AAT TT AGA TGG ATA ATG CCC CAG AG 308 59

CYP2D6 000106 CTT TCG CCC CAA CGG TCT C TTT TGG AAG CGT AGG ACC TTG 222 59

CYP2E1 000773 GAC TGT GGC CGA CCT GTT ACT ACG ACT GTG CCC TTG G 297 59

CYP3A4 017460 ATT CAG CAA GAA GAA CAA GGA CA TGG TGT TCT CAG GCA CAG AT 314 64

UGT1A6 001072 TGG TGC CTG AAG TTA ATT TGC T GGC TCT GGC AGT TGA TGA AGT A 210 60

EPHX1 000120 GGC TTC AAC TCC AAC TAC CTG GCA AGG GCT TCG GGG TAT G 184 60

GST A1 145740 TCT GCC CGT ATG TCC ACC T GCT CCT CGA CGT AGT AGA GAA GT 185 59

GST M1 000561 TCT GCC CTA CTT GAT TGA TGG G TCC ACA CGA ATC TTC TCC TCT 117 60

SULT1A1 177534 CGG CAC TAC CTG GGT AAG C CAC CCG CAT GAA GAT GGG AG 91 62

NAT1 000662 GGG AGG GTA TGT TTA CAG CAC ACA TCT GGT ATG AGC GTC CAA 128 60

NMO1 000903 TGA AGG ACC CTG CGA ACT TTC GAA CAC TCG CTC AAA CCA GC 185 61

β-actin 007393 CAC CCA CAC TGT GCC CAT C CTC CTG CTT GCT GAT CCA C 607 60


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Table 2: Reaction conditions for CYP iso-enzyme measurement

Substrate Assay Incubation Metabolite microtiter Quenching Solution Ex / Em

conc. time plate [nm]

CYP1A1/2 CEC 30 µM 90 min CHC black DPBS 390 / 460

CYP2A6 Coumarin 50 µM 60 min HC black 0.1 M TRIS (pH 9.0) 355 / 460

CYP2B6 EFC 30 µM 120 min HFC white 0.1 M TRIS (pH 9.0) 390 / 520

CYP2C8/9 DBF 10 µM 90 min Fluoresceine black 10 mM NaOH 485 / 538

CYP2D6 AMMC 10 µM 120 min AHMC white 0.1 M Tris (pH 9.0) 390 / 460

CYP2E1 MFC 10 µM 120 min HFC white 0.1 M TRIS (pH 9.0) 390 / 520

CYP3A4 BFC 100 µM 120 min HFC white 0.1 M TRIS (pH 9.0) 390 / 520


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Table 3: CYP metabolite release time-course parameter

human hepatocytes NeoHep cells

Plateau t1/2Plateau R2 Plateau t1/2Plateau R

2

[pmol metabolite/ [min] [pmol metabolite/ [min]

mg protein] mg protein]

CYP1A1/2 3022 ± 810 109.1 ± 30.0 0.7429 2683 ± 199 64.4 ± 9.9 0.8677

CYP2A6 430.1 ± 116.3 85.6 ± 34.3 0.6810 107.0 ± 24.3 77.9 ± 27.9 0.7256

CYP2B6 4199 ± 769 71.7 ± 20.0 0.7790 4055 ± 1025 102.0 ± 60.5 0.8264

CYP2C8/9 310.7 ± 73.8 100.4 ± 33.7 0.7889 277.9 ± 47.9 91.1 ± 28.5 0.6651

CYP2D6 1272 ± 257 111.7 ± 55.7 0.8231 586.3 ± 118.7 94.5 ± 27.6 0.8226

CYP2E1 3524 ± 770 89.6 ± 28.6 0.7872 1359 ± 223 67.0 ± 22.5 0.5933

CYP3A4 1262 ± 785 119.4 ± 85.5 0.8019 234.3 ± 37.0 98.5 ± 27.4 0.7242


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Table 4: Summary for time-course of metabolite conjugation data

Substrate human hepatocytes NeoHep cells

R2 Plateau half life R2 Plateau half life

[nmol substrate/ [min] [nmol substrate/ [min]

min/mg protein] min/mg protein]

AHMC 0.7506 13.47 ± 0.18 8.17 0.6584 12.32 ± 0.36 16.16

CHC 0.8303 19.73 ± 0.61 7.20 0.7921 31.21 ± 1.33 13.34

HC 0.6853 2.39 ± 0.28 12.75 0.7952 7.66 ± 0.25 16.51

HFC 0.8575 13.54 ± 1.00 18.41 0.8057 38.15 ± 1.56 28.83

Fluorescein 0.7546 8.79 ± 0.41 4.61 0.6606 10.45 ± 0.58 6.23

Resorufin 0.9026 14.13 ± 0.54 7.90 0.6246 34.19 ± 1.07 17.74

MCB 0.8868 45.93 ± 7.34 214.69 0.8630 49.10 ± 5.08 264.20


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Table 5: EC50 values for CYP1A1/2 and CYP3A4

human hepatocytes NeoHep cells

Inhibitor CYP R2 EC50 R2 EC50

[µM] [µM]

Nifedipine 1A1/2 0.9965 213.2 ± 1.36 0.9838 106.3 ± 1.28

3A4 0.9960 87.22 ± 1.13 0.9986 51.23 ± 1.05

Verapamil 1A1/2 0.9893 92.60 ± 1.32 0.9999 54.73 ± 1.01

3A4 0.9969 149.0 ± 1.17 0.9994 87.20 ± 1.05

Ketoconazol 1A1/2 0.9971 28.14 ± 1.62 0.9947 15.16 ± 1.18

3A4 0.9683 6.039 ± 1.25 0.9995 3.954 ± 1.06

Quercetin 1A1/2 0.9974 171.2 ± 1.12 0.9977 84.04 ± 1.07

3A4 0.9954 62.33 ± 1.10 0.9999 41.86 ± 1.01


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